DENDRITIC CELL FUNCTION IN THE SETTING OF CHRONIC HEPATITIS C INFECTION AND CHANGES DURING ANTIVIRAL TREATMENT Ioannis Pachiadakis UNIVERSITY COLLEGE LONDON Faculty: MEDICINE Field: HEPATOLOGY A thesis submitted to the University College London for the degree of Master of Philosophy October 2008 -1 - ABSTRACT The role of the innate immune system and in particular of dendritic cells, in the establishment of chronic hepatitis C has been studied quite extensively over the last few years but controversy remains as to whether a potential virus-induced defect in DC function leads to sub-optimal initiation of adaptive immune responses and finally to the establishment of chronic infection. In the present thesis we examined monocyte-derived DC (MDDC), generated from patients with chronic hepatitis C (CHC), and healthy subjects in order to gain a better understanding of dendritic cell functions in the setting of hepatitis C infection. We initially observed a defect in CHC patients’ DC capacity to stimulate ‘naïve’ CD4+ T-cells into proliferation. Investigating for factors potentially contributing to this defect we demonstrated that HCV genome (both positive strand HCV RNA and the replicative intermediate negative strand HCV RNA) and HCV protein products (HCV core protein) are present in DC without though any obvious correlation to DC functions. We further proccedeed to investigate for potential influence of other, host-related, parameters on DC and tested liver fibrosis, ethanol consumption, HCV viraemia levels and HCV protein products’ concentration in the serum demonstarting significant influences of HCV viraemia / HCV core serum concentration and ethanol consumption on DC functional and phenotypic output. After successful antiviral treatment (with pegylated interferon α and ribavirin) our patients restored their DC functions (allostimulatory capacity, HCV- specific immune-reactivity and IL-12production). Performing ‘cross-over’ experiments we suggest that antiviral treatment exerted its effect by improving mainly DC capacities and not effector CD4+ T-cell reactivity. We also tested plasmacytoid dendritic cell cytokine output after HCV infection and we observed a controversial effect of the virus on the Th1-skewing capacity of this DC subset. -2 - LIST OF CONTENTS Page Title page 1 Abstract 2 List of contents 3 List of tables 8 List of figures 9 Declaration 12 Acknowledgements 13 Abbreviations 14 CHAPTER1: INTRODUCTION AND REVIEW OF THE LITERATURE 15 1.1 Hepatitis C virus infection 16 1.1.1 Natural history of hepatitis C virus (HCV) infection 16 1.1.2 Biology of HCV 17 1.1.2.1 Virion structure 17 1.1.2.2 HCV proteins 18 1.1.3 HCV lifecycle 24 1.1.3.1 Entry receptors 24 1.1.3.2 Intracellular lifecycle of HCV mechanisms of viral replication 25 -3 - 1.2 Role of the immune system in the control of HCV infection 28 1.2.1 Innate immune response.The first line of defence 28 1.2.2 Adaptive immune responses 29 1.3 The role of dendritic cells in the generation of adaptive immune responses against viral infections 34 1.3.1 Dendritic cells 34 1.3.2 Dendritic cell subsets 36 1.3.3 Dendritic cell activating ligands 41 1.3.3.1 Pattern recognition receptors 41 1.3.3.1.1 TLR family 43 1.3.3.1.2 C-type lectin family 45 1.3.4 Signalling pathways involved in dendritic cell physiology 48 1.4 Dendritic cells in the setting of chronic HCV virus infection 51 1.4.1 Viruses exploit DC to establish chronicity 52 1.4.2 Role of DC in Flavivirus infection 55 1.4.3 The role of DC in HCV infection 55 1.4.3.1 How does HCV bind to DC? 55 1.4.3.2 Does HCV replicate in DC? 58 1.4.3.3 Effects of chronic HCV infection on DC function 60 1.4.3.3.a Capacity to stimulate T-cells 60 1.4.3.3.b Surface marker expression on infected DC 62 -4 - 1.4.3.3.c Cytokine secretion 63 1.4.3.3.d NK cell stimulation 64 1.4.4 Potential mechanisms HCV-induced disruption of DC function 65 1.5 AIMS OF THE STUDY 69 CHAPTER 2: MATERIALS AND METHODS 70 2.1 Materials 71 2.1.1 Clinical material – patients 71 2.1.2 Materials 71 2.2 Methods 72 2.2.1 Cell cultures 72 2.2.2 Cell separation and storage 73 2.2.3 Generation of monocyte-derived dendritic cells (MDDC) 75 2.2.4 Isolation of CD4+ T-cells 76 2.2.5 CD40-ligand(L) – expressing human lymphoid cells 77 2.2.6 Plasmacytoid dendritic cell (PDC) isolation 77 2.2.7 Cytokine assays 79 2.2.7.1 Enzyme-linked immunospot (ELIspot) assay 79 2.2.7.2 Enzyme-linked immusorbent assay (ELISA) 81 2.2.8 Cell proliferation assays 83 2.2.9 Total RNA extraction from PBMC and MDDC 85 2.2.9.1 Using the QIAGEN RNeasy Minikit 85 -5 - 2.2.9.2 Using a Trizol chloroform-isopropanol precipitation protocol 86 2.2.10 Total RNA extraction from plasma 86 2.2.11 Real-time RT-PCR for the detection of HCV RNA in PBMC and MDDC 87 2.2.11.1 Real-time RT-PCR assay 87 2.2.11.2 Standardisation/normalisation of the real-time RT-PCR 90 2.2.12 HCV RNA quantitation in patients’ plasma and HCV genotype determination 92 2.2.13 Nested RT-PCR-nucleic acid hybridisation (RT- PCR-NAH) for the detection of HCV-specific sequences in PBMC and MDDC 93 2.2.14 Surface marker expression on MDDC and PBMC 94 2.2.15 Intracellular and plasma HCV Core antigen quantitation 95 2.2.16 Antiviral treatment 96 2.2.17 Liver biopsy 97 2.2.18 Statistical analyses 97 CHAPTER 3: DENDRITIC CELL FUNCTION IN THE SETTING OF CHRONIC HCV INFECTION 105 3.1 Introduction 106 3.2 Results 110 3.2.1 MDDC allostimulatotry capacity 110 3.2.2 Assessment of factors potentially interfering with DC function in the setting of chronic HCV infection 111 -6 - 3.2.2.1 Presence of HCV-specific genomic sequences in MDDC from chronically infected patients (CHC-DC) 111 3.2.2.2 HCV RNA levels in patients’ plasma 116 3.2.2.3 Ethanol consumption 117 3.2.2.4 HCV Core protein levels in MDDC lysates 118 3.2.2.5 HCV Core protein levels in patients’ plasma 119 3.2.3 Effect of antiviral treatment on DC functions 120 3.2.4 ‘Cross-over’ experiments 122 3.2.4 Plasmacytoid dendritic cells 124 3.3 Discussion 129 CHAPTER 4:SUMMARY AND CONCLUSIONS 160 4.1 Summary 161 4.2 Conclusions 167 BIBLIOGRAPHY 169 PUBLICATIONS – PRESENTATIONS 197 -7 - LIST OF TABLES CHAPTER 1: Table 1.1 TLR-ligands 44 Table 1.2 Disruption of DC functions by viruses 54 CHAPTER 2: Table 2.1 Patients’ characteristics 99 Table 2.2 Maturation of MDDC with 0.1μg/ml LPS Representative FACS data from a single patient (GS) 100 Table 2.3 Real-time TaqMan PCR reaction mixture 101 Table 2.4 Determination of the analytical sensitivity of real-time RT-PCR using a dilutional series of the HCV 5’UTR RNA transcripts from 10 to 107 molecules and tested five times, each in duplicate 102 Table 2.5 Determination of the detection limit of the real- time RT-PCR assay 103 Table 2.6 Reaction mixture used for real-time RT-PCR for the detection of ‘house-keeping’ GAPDH genomic sequences in MDDC lysates 104 CHAPTER 3: Table 3.1 HCV RNA detection by TaqMan real-time RT- PCR in cell lysates (MDDC and PBMC) and in patients’ plasma 136 -8 - Table 3.2 Total RNA recovery from MDDC lysates and HCV RNA positive strand and negative strand detections 137 Table 3.3 Patients’ characteristics for the ‘cross-over’ experiments 138 Table 3.4 Correlation of the presence of positive-strand HCV RNA and negative-strand HCV RNA in MDDC lysates to: cell-surface marker expression (a. and c. respectively) and functional parameters of MDDC (b. and d. respectively) 139 LIST OF FIGURES CHAPTER 3: fig.3.1 Allostimulatory capacity of LPS-matured DC from 30 patients with chronic HCV infection (Patients) and LPS-matured DC from 12 non-infected healthy individuals (Controls) 140 fig.3.2.a HCV RNA plasma levels and effect on CD40 expression on immature DC 141 fig.3.2.b HCV RNA plasma levels and effect on CD80 expression on immature DC 142 fig.3.2.c HCV RNA plasma levels and effect on CD40 and CD80 expression on immature DC. Scatterplot of raw data corresponding to data presented in figures 3.2.a and 3.2.b 143 -9 - fig.3.3 Effect of ethanol (EtOH) consumption (units/week) on IL-12p70 production 144 fig.3.4.a Effect of HCV core plasma levels on DC allostimulatoty capacity (MLR assay with LPS- matured DC) 145 fig.3.4.b HCV core plasma levels and effect on CD83 expression on LPS-matured DC surface 146 fig.3.4.c HCV core plasma levels and effect on CD86 expression on immature DC surface 147 fig.3.4.d Effect of HCV core plasma levels on DC allostimulatoty capacity (MLR assay with LPS- matured DC). Scatterplot of raw data corresponding to data presented in figure 3.4.a 148 fig.3.4.e HCV core plasma levels and effect on CD83 expression on mature DC and CD86 expression on immature DC. Scatterplot of raw data corresponding to data presented in figures 3.4.b and 3.4.c 149 fig.3.5.a Autologous CD4+ T-cell response, tested with IFN-γ Elispot assay, for ‘good’ and ‘poor’ responders to antiviral treatment, with DC pulsed with HCV core protein 150 fig.3.5.b Autologous CD4+ T-cell response, tested with - 10 - IFN-γ Elispot assay, for ‘good’ and ‘poor’ responders to antiviral treatment, with autologous DC pulsed with HCV NS3 151 fig.3.6 Allostimulatory capacity in ‘good’ and in ‘poor’ responders to antiviral treatment 152 fig.